Publication Details

Felletti, M., Jehle, S., Wang, Y., Ozawa, K., Jergic, S., Otting, G., Emsley, L., Lesage, A., Dixon, N. & Pintacuda, G. (2012). Ultra fast MAS solid-state NMR studies of protein-protein interactions in the bacterial replisome. The Febs Journal, 279 (1), 472-472.


Replication of genomic DNA in bacteria involves multiple stable and transient interactions among protein subunits constituting the replisome. Here we show how ultra-fast magic angle spinning (MAS) solid-state NMR (ssNMR) and 1H-detection on either deutereted or fully protonated samples, can give new information about protein-protein interactions in the E. coli replisome. We focused on two fundamental interactions: the first one between the C-terminal domain of the single-stranded (ss) DNA binding protein and its own DNA-binding site, and the second one between the DNA polymerase III subunits a and s. SSB (a tetramer of 79 kDa) has a structurally well-defined ssDNA binding domain (OB-domain) and an intrinsically disordered C-terminal (Ct) domain. Its extreme Ct acidic motif is known to mediate the binding of SSB to different DNA processing enzymes and scaffold proteins. ssNMR provides, for the first time, residue-specific evidence for interaction, in multiple heterogeneous conformations, of SSB-Ct with its own ssDNA-binding site. In particular we compared spectra from the native protein and from a deletion mutant lacking the extreme Ct. This interaction acts as a switch that directs recruitment of SSB-binding proteins specifically to SSB only when it is bound to ssDNA. Moreover we investigated the interaction between the C-terminal domains of a (aCTS 22.5 kDa) and s (sC16 16 kDa). The structure of the complex is so far unknown. We used cell free protein synthesis to produce aCTS in the presence of sC16. We analyzed two different samples in which each the interacting partners is separately 2H,13C,15N labelled. The complete resonance assignment of the proteins in the complex opens the way to mapping the protein-protein contacts and to the determination of the overall structure

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